Cloning Research

Taken from "A Review of Cloning in the Horse" by Dr. Katrin
Hinrichs

The Equine Embryo Laboratory is at the forefront of cloning
research. Having successfully cloned 7 donors for 15 live foals,
the staff continues to do research so that their cloning efforts
can be used to benefit society as a whole. Cloning can be used to
produce breeding animals to help preserve valuable equine
genetics.

Introduction

Equine cloning has been discussed in the popular press since the
birth of the first cloned equids (three mules and one horse) in
2003. In general, interest has been centered on whether or not the
cloned offspring will be normal, how closely they will resemble the
donor animal, and what cloning may be used for within the industry.
Although equine cloning is still in its infancy, sufficient
information is available from other species and from the few equine
clones already produced to allow us to start answering these
questions.

Technique

The principle of cloning is relatively simple. The chromosomes
of a cell from the donor animal are transferred into the cytoplasm
of an egg, and the egg is signaled to develop an embryo. The cells
from the donor animal are typically grown from a small sample of
subcutaneous connective tissue. At the laboratory, the tissue is
placed into culture, and fibroblasts are grown from it onto the
culture dish. The fibroblasts will proliferate until they cover the
bottom of the plate and they may be resuspended in medium and used
to "seed" additional dishes. After a sufficient number of cells are
obtained, the cells are typically frozen to be used at a later
time.

Oocytes used for cloning may be harvested from the dominant
pre-ovulatory follicles of live mares or they may be obtained by
maturing immature oocytes in vitro. The donor cell is then combined
with the enucleated oocyte either by electrofusion or by directly
injecting the cell into the cytoplasm of the oocyte. The recombined
oocyte is activated to stimulate embryonic development; this is
typically done by triggering calcium oscillations within the oocyte
that mimic those that occur at fertilization.

After the recombined oocyte has been activated, it may be
transferred surgically to the oviduct of a recipient mare, or it
may be cultured in vitro to the blastocyst stage for transfer
directly to the uterus of a recipient mare as for standard embryo
transfer.

How Closely will the Clone Resemble the Donor

A variety of factors will affect the degree of similarity
between the cloned offspring and the original donor, but only two
are actually related to the cloning procedure. Epigenetic changes
compatible with a viable foal may still cause the gene function of
a cloned foal to differ somewhat from that of the donor; therefore,
the foal may potentially be shorter or taller, have more or less
bone, etc. than did the donor animal. The second cloning-related
potential cause of differences between the clone and the donor
animal is related to mitochondria.

If the cloned embryo was cultured in vitro before transfer to
the recipient mare, in vitro culture itself has been shown to cause
differences in neonatal size and other phenotype differences in
other species.

Other potential causes of differences between the cloned foal
and the donor would be seen in any transferred embryo; however,
they will be seen in any transferred embryo; however, they will be
more obvious in cloned foals because the expected phenotype is
known.

These variations in phenotype and in mitochondrial genotype will
be useful in identifying individual cloned offspring that are
produced from the same genetic donor. The possibility of phenotypic
variation in cloned offspring as well as possible health problems
associated with cloned neonates makes it unlikely that the cloned
offspring will perform at the same level as the donor animal.

How Will Cloning Affect the Industry

Cloning is most accurately viewed as a method for genetic
banking, similar to freezing semen or oocytes so that progeny may
be obtained from a genetic line after the original horse is no
longer fertile or is deceased. However, cloned horses are currently
not eligible for registration with most breed registries in the
United States.

Even in the United States, cloning is currently applicable to
horses in which the value of the progeny does not depend on
registration with a breed association. Thus, cloned animals may
produce progeny that could compete in dressage, jumping,
cross-country, polo, cutting, reining, or other events.

The possibility exists with cloning for misuse and manipulation,
and it is difficult to predict the range of these potential
problems. The cloned animals themselves will be different from each
other and from the original donor by their markings and their
mitochondrial genotype. However, not only is cloning inefficient
and costly, but it is also unlikely to produce a champion of the
same quality horse because of the various factors potentially
affecting the performance of cloned foals.

Can the progeny of cloned horses be differentiated from the
progeny of the other horses? Progeny of cloned mares will be
different from progeny of the original mare by their mitochondrial
DNA. However, progeny of cloned stallions may not be different from
progeny of the original stallion. Substitution of semen from a
clone for semen from the original stallion would need to be
monitored by evaluating the mitochondrial DNA from the semen
sample. The small number of mitochondria in a spermatozoon presents
some problems for efficient genotyping; this is an area that is
currently under investigation at our genetics laboratory at Texas
A&M University.

Summary

Equine cloning is possible today, and its value to the industry
will be determined over the next few years. Cloning should be
viewed as a method for producing a breeding animal rather than as a
means to "duplicate" a performance horse. To the equine
practitioner, cloning provides a procedure that may be offered to
clients to preserve valuable genetics in the face of reproductive
problems that in the past were insurmountable.